T helper cells in lymphoid tissues adopt multiple cell fates that regulate inflammation. At the G-I mucosa, their dominant phenotype is of Treg cells. Mucosal adjuvants, by overriding the tolerant impact of mucosal Treg cells, are unique tools for dissecting mucosal regulation of immunity and inflammation. Our preliminary data indicate that cholera toxin (CT) and related compounds induce Th17 cell differentiation via a cAMP-dependent and an IL-6/TGFb-independent pathway. To elucidate the mechanisms by which adenylyl cyclase toxins and other related cAMP-inducing compounds mediate mucosal adjuvanticity and to explore their potential inflammatory impact in vivo, we propose in SA-1 to evaluate whether oral CT administration enhances Th17- promoting DC and negatively regulates Treg-promoting DC. We will explore the role of cAMP and retinoic acid in this process and will measure and characterize the subsequent Th-cell response provoked by different MLN DC subsets. In SA-2, we will explore the role of CGRPb produced by CT-activated DC in a mucosal Th17 response and will dissect CT-induced differentiation of Th17 cells in vitro and in vivo using various ko mice and pharmacological approaches in wt animals and we will assess the ability of these manipulated mice to mount mucosal immune responses to oral OVA/CT immunization in vivo. Dissecting the impact of CT and other cAMP-inducing compounds on mucosal DC maturation will enrich our knowledge related to the generation of mucosal Th responses. By dissecting the lineage stability of the CT- induced mucosal Th17 cells, their effector function, lineage commitment, trafficking and their interactions with other Th cells, we will increase our understanding of the regulation of inflammation vs. immunity at mucosal sites. These data will increase our knowledge related to the induction of immunity and the regulation of inflammation at mucosal sites. We believe that our studies will provide a new foundation for the design of novel mucosal adjuvants and/or modulators of mucosal inflammation (Celiac and IBD) and may identify adjuvants with a better safety profile than CT for future clinical use. In the proposed studies we will explore the cellular and molecular pathways by which cholera toxin mediates its mucosal adjuvanticity.